Flow control in high performance liquid chromatography

ABSTRACT

A method for controlling the flow of liquid in a high performance liquid chromatography apparatus. The method includes operating a pump, measuring the liquid pressure downstream of the pump, measuring the liquid flow rate downstream of the pump, and controlling the operation of the pump. In the method, it is automatically determined whether the pump is controlled to achieve a desired pressure or controlled to achieve a desired flow rate. Fuzzy logic can be applied in the method to determine the switch between the control modes.

The present application relates to flow control in liquid chromatographyand in to particular reverse phase HPLC (High Pressure (or Performance)Liquid Chromatography) devices and methods.

BACKGROUND OF THE INVENTION

HPLC is used ubiquitously for both analytical and preparative separationof molecules in diverse areas such as research, development andproduction in chemistry, pharmaceuticals, biotechnology, fundamentallife science studies, etc.

One particular use of HPLC is in the field of proteomics, i.e. the studyof the entire protein complement of a cell or tissue sample whereproteolytic fragments of proteins (e.g. peptides) are separated by HPLCprior to detection by mass spectrometry. Since the samples beinganalyzed in proteomics experiments are typically very complex andavailable in only very low quantities, it is frequently a challenge toobtain sufficient sensitivity and analysis speed.

Sensitivity is obtained by using low flow rates for the mobile phase incombination with nano-bore columns (i.e. columns of narrow innerdiameter). This approach however often leads to back pressures that arein excess of the instrumental system tolerances, which may cause systemfailure and also in many cases requires prolonged analysis times, whichin turn leads to poor duty cycles for the overall LC-MS analysis. Theseback pressure and duty cycle problems affect many other applicationareas beyond proteomics as well.

In known proteomics, typical experimental HPLC conditions and parameterscurrently are:

Flow range: 100 nL/min-500 nL/min (also called nano-LC)

Pressure range: 50 atm.-600 atm.

Liquid phases are typically:

A-buffer: Mainly aqueous, often acidified and containing additives, withno or low organic content

B-buffer: Mainly organic, often acidified and containing additives, withno or low water content

Gradients are typically: From no or low percentage B-buffer to highpercentage B-buffer in 5 to 600 minutes. Standard gradients could befrom 5% B to 90% B in 30 minutes.

Stationary phases are typically:

Beads of various materials, often highly porous, diameters of 1.5 μm-5μm, hydrophobic coating of hydrocarbons (C8 or C18) with chemicalend-cap (non-hydrocarbon functional group)

Column sizes are typically:

ID: 25 μm to 250 μm

Length 1 cm to 200 cm

The operation of reverse phase nano-HPLC (or rather, the execution of ananalysis cycle) can be separated into several chronologically distinctsteps:

-   -   1. Loading of sample; either from an auto-sampler or by manual        injection with a syringe into a sample loop. Sample volumes in        nano-LC applications are typically 0.5 μL to 10 μL (but can be        10 times larger or smaller than this range).    -   2. Re-location of the loaded sample from the loop and onto the        column.    -   3. De-salting of the immobilized sample by a volume of buffer        (typically 100% A-buffer, i.e. with little or no organic        solvent). This volume is typically 1.5 to 3 times larger than        the volume from which the sample was loaded (e.g. a 5 μL sample        injection would be de-salted with 7.5 μL to 15 μL of buffer) but        the de-salting buffer volume can also be much larger or smaller        than this range.    -   4. The elution and separation step. This is either done as        isocratic elution (i.e. where the buffer composition remains        constant during the elution step) or as a gradient where an        continuously increasing ratio of organic is used.    -   5. A column cleaning step where all analytes of interest have        been eluted but an extra high concentration of organic solvent        is applied to the column in order to remove strong-binding        molecules prior to the next analysis cycle. Such molecules would        typically include pollutants such as organic polymers,        surfactants, and very large bio-molecules that would interfere        with subsequent separations if allowed to accumulate on the        solid phase material.    -   6. A column re-equilibration step wherein the high-organic        buffer inside the column is displaced with pure A-buffer such        that the solid phase material can bind the next sample that is        loaded.

Steps 5 and 6, or just step 6, can also be performed as the first stepinstead of being the last step(s) of the cycle. The above six steps ofthe operation can also be classified differently and be sub-divided intoyet other steps.

The flow rates may change widely from step to step and also within eachstep. It is possible to regulate the flow and obtain workingchromatography by regulating pump speeds such that the system strives toachieve a requested back pressure. This method of “pressure regulatedflow control” is unfortunate inasmuch as it can lead to highly variableand badly controlled flow rates. Hence this control method is now veryrarely used, and a more common way to regulate the pump speeds is bycontrolling for the actual absolute flow rates as measured by flow ratesensors somewhere downstream of the pump. Hereinafter, these twodifferent modes of pump regulation will be referred to as “pressurecontrol” and “flow control”, respectively.

In the prior art the above steps, that encompass one analysis cycle, areeither regulated by pressure control or flow control, with flow controlbeing by far the most commonly used regulatory method. On some prior artsystems, the pressure and flow during analysis execution are notregulated at all.

The detection efficiency by means of electrospray mass spectrometry isdependent on the analyte concentration at the time of ion formation, sothe best sensitivity is achieved when analytes are eluted in volumesthat are as small as possible.

This is done by using columns of narrow inner diameters and low flowrates. The narrow inner diameter of columns causes a large resistance tothe flow of the mobile phase. Although the columns are filled withstationary phase material, the flow through the columns and the LCtransfer lines is largely following Poiseulle's Law concerning thelaminar flow of a Newtonian fluid in circular tubes. The Poiseulleequation states that the flow resistance is proportional to the lengthof the column and the viscosity of the mobile phase, while it is alsoinversely proportional to the column radius to its fourth potential.Hence, the resistance increases as columns become longer and narrower,especially during times of the analysis where the organic solvent ratioof the mobile phase is low. The “back-pressure” of the LC system ishereinafter considered as being the pressure delivered by the LC pumpsin order to obtain a desired flow. Every LC system has an upper limit ofits back-pressure above which either safety mechanisms will turn off thepumps (thereby halting the analysis) or system components will begin tofail (e.g. valves, seals, and fittings will break or leak).

In any case, it is currently necessary to take extraordinary care toensure the back pressure does not exceed the system limitations at anypoint of the analysis cycle. For that reason, it is customary to performa few “dry-runs” and monitor the back-pressure whenever a new column, ora new mobile phase, or a new method is deployed. Then the analysisparameters are typically adjusted such that the maximum back-pressurelies well below the system limits, including an extra safety marginsince it is an established problem that columns exhibit increasingback-pressure contributions over time (owing to the accumulation ofparticulate debris at the column front and in-between column beads). Thesystem's back-pressure limitation typically does not put any severeconstraints on the execution of analyses during the part of theexecution where the analytes are eluted (during the gradient), sincethat is typically done using very low flow rates (which in turn leads toa low back pressure).

However, in order to save considerable amounts of analysis time, it istypically advantageous to significantly increase the flow rates duringthe parts of the analysis cycle where: i) a sample is loaded onto acolumn; ii) the column or sample is being de-salted; and iii) the columnis being re-equilibrated. During such parts of the analysis cycle, thesystem back pressure limitation poses severe constraints, andexperimental parameters must (with current technology) be selected witha substantial safety margin, thereby leading to loss of analysis time.Otherwise, the execution will fail with substantial frequency, therebyleading to loss of samples and/or instrument damage and/or subsequentloss of time (since samples have to be re-analyzed).

Typical prior art nano liquid chromatography systems have upperback-pressure limits of around 5,000 PSI (340 bar), whereas some systemsare designed to remain operational at over 10,000 PSI and are calledUltra-high performance liquid chromatography systems (UPLC). UPLCs arehowever designed to take advantage of columns that in general elevatesthe back pressure, so the general problem remains and one must stilltake active steps to avoid an “over-pressure” situation during all partsof the analysis cycle.

DISCLOSURE OF THE INVENTION

On this background, it is an object of the present application toprovide a method for controlling fluid flow in a liquid chromatographyapparatus that overcomes or at least reduces the drawbacks indicatedabove, i.e. a method that alleviates the problems of excessive backpressures and long duty cycles.

This object is achieved by providing a method for controlling fluid flowin a liquid chromatography apparatus, the method comprising driving saidflow with a pump, controlling said fluid flow by either measuring thepressure of said liquid and in response thereto adjusting the output ofsaid pump to achieve a target fluid pressure, or measuring the fluidflow of said liquid and in response thereto adjusting the output of saidpump to achieve a target fluid pressure, and automatically switching orbalancing between pressure based fluid flow control and flow rate basedfluid flow control.

Thus according to the invention there is switching between the two modesof control or there can be a mix of the two control modes in anautomatically determined balance. By automatically switching orbalancing between the pressure control mode and the fluid flow ratecontrol mode, the more optimal control mode for the actual circumstancescan be used.

The target fluid pressure and/or the target flow rate can be variableduring the execution of the method.

The method may further comprise involving fuzzy logic algorithms fordeciding when to switch between said pressure based fluid flow controland flow rate based fluid control.

By applying fuzzy logic, it becomes possible to concurrently optimizethe analysis speed, while greatly improving both the ease-of-use of theapparatus and the certainty of flawless analysis execution.

Fuzzy logic is a computationally straightforward and very robust way toobtain a working regulatory mechanism that works well both understandard conditions as well as under extreme conditions and duringunfortunate events such as e.g. instant line blockage.

The liquid chromatography apparatus can be operated in an analysis cyclethat involves a plurality of chronological steps that have differentfluid flow requirements, and one or more of said steps beingpredetermined to be operated with either flow rate based control orpressure based control.

One or one or more of said steps may not be predetermined to be operatedwith a particular one of the flow rate based control or pressure basedcontrol mode, and the control mode in these steps is determined on thebasis of measured parameters relating to operation of the liquidchromatography apparatus.

The fuzzy logic can have the current flow rate and the current pressuredownstream of the pump as the input parameters.

The fuzzy logic output parameter can be the pump speed.

The fuzzy logic can be arranged and divided into a plurality ofoverlapping fuzzy sets. The fuzzy logic sets can be configured to giveboth course- and fine-grain regulation characteristics.

The output variable of the fuzzy logic can be arranged into a pluralityof discrete singleton outputs, which are subsequently defuzzified into acontrol value by a center of gravity method.

The fuzzy variables can be featured in a plurality of rules, aiming tooptimize flow, while at the same time ensuring that a maximum pressureis not exceeded.

Rules pertaining to pressure regulation can be prioritized by rulefactors, so that pressure regulation will take precedence over any flowoptimization, when the regulator operates in that section of the inputspace.

The fuzzy logic flow control may comprise an iterating loop that takesthe sensor readouts, supplies them to a fuzzy logic engine and has thefuzzy logic engine re-evaluate its flow output.

The output difference maybe calculated relative to the previous outputvalue and said difference is clamped into a specific range, added to theprevious output value, and supplied to the pump as a new pump speed.

The object above can also be achieved by providing a liquidchromatography apparatus comprising at least one pump, a flow ratesensor downstream of said pump, a pressure sensor downstream of saidpump, and a pump controller coupled to said pump, to said flow ratesensor and to said pressure sensor, said pump controller beingconfigured to automatically switch between or balance pressure basedpump control and flow rate based pump control.

Preferably, the pump controller includes a fuzzy logic engine.

Object above can also be achieved by providing a computer readablemedium including at least computer program code for controlling theoperation of a pump and program code for automatically switching betweenor balance a pressure control mode and a flow rate control mode.

Preferably, the computer readable medium includes program code forapplying fuzzy logic.

Further objects, features, advantages and properties of the method,apparatus and computer readable medium according to the invention willbecome apparent from the detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following detailed portion of the present description, theinvention will be explained in more detail with reference to theexemplary embodiments shown in the drawings, in which:

FIG. 1 is a diagrammatic scheme of a liquid chromatography apparatusaccording to an embodiment of the invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In the following detailed description, the apparatus, the method and thecomputer readable medium according to the teachings of this invention inthe form of a liquid chromatography apparatus will be described by theembodiments.

FIG. 1 illustrates an embodiment of an apparatus according to anembodiment of the invention in the form of a high performance liquidchromatography apparatus.

The high performance liquid chromatography apparatus which is generallydenoted with 1 is provided with a nano-liter syringe pump 10 that uses adirect drive crystal (sapphire) piston for delivering high pressuresolvent A and a nano-liter syringe pump 12 that also uses a direct drivesapphire piston but in this case for delivering high pressure solvent B.

The syringe pump 10 is driven by stepper motor 14 to deliver pressure tosolvent A (A-buffer) and the syringe pump 12 is driven by a steppermotor 16 to deliver pressure to solvent B (B-buffer). The outlet/inletof the syringe pump 10 is connected to a first valve 18 and theoutlet/inlet of the syringe pump 12 is connected to a second valve 20.Both first valve 18 and second valve 20 has two positions that areachieved by a 60° rotation. The little circles represent the six portsof the valve (not all ports are in use) and the thick black parts of thelarger circle represent fluid connections whilst the white parts of thelarger circle illustrate that there is no fluid connection between theports concerned.

In the shown position first valve 18 connects the syringe pump 10 toconduit 29 that leads the fluid from the syringe pump towards thechromatography column 50. This position is e.g used during a sample run.

In the other of the two positions (not shown) the first valve 18connects the syringe pump 10 to a canister 15 that contains solvent A.This position is used for refilling the syringe pump with solvent A.

In the shown position second valve 20 connects the syringe pump 12 toconduit 31 that leads the fluid from the syringe pump towards thechromatography column 50. This position is e.g. used during a samplerun.

In the other of the two positions (not shown) the second valve 20connects the syringe pump 12 to a canister 17 that contains solvent B.This position is used for refilling the syringe pump with solvent B.

A pressure sensor 24 measures the pressure in the liquid downstream ofpump 10 and a pressure sensor 26 measures the pressure in the liquiddownstream of pump 12. A flow sensor 28 in-line with conduit 29 measuresthe fluid flow rate of solvent A and a fluid flow sensor 30 measures thefluid flow rate of solvent B in conduit 31.

A controller 22 receives the sensor information from pressure sensor 24,pressure sensor 26, flow rate sensor and from flow rate sensor 30. Thecontroller 22 is coupled to the stepper motor 14 and to the steppermotor 16 and issues a control signal to the stepper motor 14 and to thestepper motor 16. These control signals determine the speed of thestepper motors 14,16.

Conduit 29 and 31 (buffer lines) are joined at a narrow Tpiece/connector 32. The outlet of the T-connector 32 is coupled to athird (auto sampler) valve 34. The third valve 34 is similar to thefirst and second valves 18,20, in that it has six ports (all ports arein use) and two positions that are obtained by a 60° rotation. In theshown position in the third valve 34 connects the outlet of the T-pieceto the conduit 43 that leads to column 50. In this position the thirdvalve 34 also connects a sample plate (or microtiter plate) 38 with thesamples to one end of a sample collecting coil 36 and connects the otherend of sample collecting coil 36 to a syringe pump 40 that is driven bystepper motor 42.

This position of the third valve 34 is used to aspirate a sample fromthe sample plate 38 into the sample coil 36 by withdrawing the piston ofthe syringe pump 40 until the sample has moved into the sample coil 36.In the other of its two positions (not shown) the third valve 34connects the outlet of the T-connector 32 to one end of the samplecollecting coil 36 and the other end of the sample collecting coil 36 toconduit 43 that leads to the pre-column 46 and the column 50.

A connector 44 couples of the conduit 43 to the valve 34. Conduit 43includes a pre-column 46, a T-connector 48 and the (main) column 50. Onebranch of the T-connector 48 is connected to a waste conduit 51 thatincludes a fourth valve 52, that has two positions and six ports (onlytwo ports are in use) and opens and closes the connection of conduit 51to a waste container 54 by a 60° rotation of the valve.

Components 46, 48, 51, 52, and 54 are omitted in another embodiment ofthe chromatographic device (not shown). In this alternative embodimentthe sample is relocated from the sample coil 36 directly onto the column50 instead of onto the pre-column 46.

For practical reasons (i.e. to avoid that the drawing will be clutteredwith non-essential information) the connections between the controller22 and the third valve 34, the fourth valve 54 and the stepper motor 42have not been shown in FIG. 1. It is understood that the first, second,third and fourth valve 10, 12, 34, 52 are controlled by the controller22 and operate automatically without the interference of an operator oruser. The controller 22 is configured to execute the complete analysiscycle automatically, based on pre-programmed instructions and commandsentered by an operator.

The operation of reverse phase nano-HPLC (or rather, the execution of ananalysis cycle) is in this embodiment of the invention separated intoseveral chronologically distinct steps:

-   -   1. Loading of sample; either from the sample plate 38 or by        manual injection with a syringe into the sample loop 36. Sample        volumes in nano-LC applications are typically 0.5 μL to 10 μL        (but can be 10 times larger or smaller than this range).    -   2. Re-location of the loaded sample from the sample loop 36 and        onto the pre-column 46 or the column 50 as the case may be.    -   3. De-salting of the immobilized sample by a volume of buffer        (typically 100% A-buffer, i.e. with little or no organic        solvent). This volume is typically 1.5 to 3 times larger than        the volume from which the sample was loaded (e.g. a 5 μL sample        injection would be de-salted with 7.5 μL to 15 μL of buffer.    -   4. The elution and separation step. This is either done as        isocratic elution (i.e. where the buffer composition remains        constant during the elution step) or as a gradient where an        continuously increasing ratio of organic is used.    -   5. A column cleaning step where all analytes of interest have        been eluted but an extra high concentration of organic solvent        is applied to the column in order to remove strong-binding        molecules prior to the next analysis cycle. Such molecules would        typically include pollutants such as organic polymers,        surfactants, and very large bio-molecules that would interfere        with subsequent separations if allowed to accumulate on the        solid phase material.    -   6. A column re-equilibration step wherein the high-organic        buffer inside the column is displaced with pure A-buffer such        that the solid phase material can bind the next sample that is        loaded.

Steps 5 and 6, or simply step 6, can also be performed as the first stepinstead of being the last step of the cycle. These steps can also beclassified differently and be sub-divided into yet other steps.

In prior art, the analysis method (i.e. the method according to whichthe analysis cycle is executed) is controlling the pump speed based onflow rate measurements from the two flow sensors during all six steps ofthe analysis cycle defined above.

According to the present embodiment of the invention, the conventionalflow rate control algorithm in the controller 22 has been replaced inall steps except step 4 (the gradient, which is still strictly regulatedfor flow rate and A/B buffer ratio) and 5 (which is executed as anintegral part of the gradient and hence uses the same controlalgorithms) by a control method that switches automatically between flowrate control and pressure control.

In an embodiment, the controller 22 has been provided with a fuzzy logicengine. In the fuzzy logic engine operates with mathematical logiatechnology, which operates with approximate reasoning based on logicallydefined sets.

Where classical bivalent predicate logic incorporates only discrete setrelationships, resulting in either-or Bayesian inferences, fuzzy logicenables a more gradual view of set relations, with the ability toproduce regulator inferences with diversified multi-objective responses.The behavior of an inference engine based on fuzzy sets—a fuzzy logiccontroller—is specified by fuzzy logic rules that share the syntax ofconventional Bayesian logic, but they operate on input and output fuzzysets, which semantically represent approximate reasoning and subsequentcontrol. Input variables are converted into fuzzy set membership values,are handled by the fuzzy control rules, resulting in several piecewiseoutput sets, which are finally “defuzzified” into a singular real outputcontrol value.

According to an embodiment a standard implementation of a fuzzy logiccontroller is described by the standardized (IEC 1131-7) Fuzzy ControlLanguage (FCL), and in the present embodiment, with the followingconfiguration:

-   -   Two input variables: the current flow and the current pressure,        arranged and divided into 9-10 overlapping fuzzy sets, designed        to give both course- and fine-grain regulation characteristics.    -   One output variable: the control flow given to the pump,        arranged into 9 discrete singleton outputs, which are        subsequently “defuzzified” into a control value by the “Center        of Gravity” method (for singleton sets).

These fuzzy variables are featured in 12 rules, aiming to optimize flow,while at the same time ensuring a maximum pressure is not exceeded.Rules pertaining to pressure regulation are prioritized by rule factors,so that pressure (down-)regulation will take precedence over any flowoptimization, when the regulator operates in that section of the inputspace. This allows for seamless multi-objective optimization.

In an embodiment of the specific implementation of the fuzzy logiccontrol as used during the analysis cycle is as follows:

The fuzzy logic controller 22 is a specialized case of a conventionalpump flow controller, which is a pluggable algorithm (known as astrategy) that alters the behavior of the pump 12, 14 depending on itsimplementation. For the pump (as a software device), the controllerimplementation is completely opaque, i.e. the pump is independent of howit works and is implemented. The controller has access to the readoutsof the flow and pressure sensors downstream of the pump.

During the analysis cycle, different flow controllers (including thefuzzy logic controller) are plugged into the pump device, thereby takingcontrol of how sensor feedbacks are interpreted and processed, and inturn, how pump flow is regulated. According to an embodiment of theinvention, the specific controller used in each analysis step ispreprogrammed.

In the analysis steps where fuzzy logic flow control is used (steps 2, 3and 6), an iterating loop will take the sensor readouts (flow rate andpressure), supply them to the fuzzy logic engine and have it re-evaluateits flow output. The output difference is then calculated relative tothe previous output value. This difference is clamped into a specificrange, added to the previous output value, and supplied to the pump as anew flow rate (pump speed/speed of the stepper motor). Clamping isincluded to protect against fast changes in output flow rate, i.e.changes that are greater than the mechanical/electrical capabilities ofthe pump. The output flow rate is e.g. recalculated every 200 ms.

Steps 2, 3 and 6 are normally only restricted by system and/or columnpressure limits, so for ease of use, the researcher (user/operator) isallowed to only specify a pressure limit and volume, but leave the flowrate parameter undefined. In this way the system can complete thesesteps at a flow rate as high as possible, i.e. as quickly as possible,without the researcher having to worry about a suitable flow rate tostay within pressure limits. On the other hand, if so desired, a flowrate can be specified by the operator, and the controller 22 will thentry to deliver this particular flow, but still ensure the back pressurestays within the specified limits.

The fuzzy engine in the controller 22 uses rules featuring course anddrastic changes when operating far from the typical flow setpoint value.When it operates near the setpoint, however, more fine-grained anddetailed rules take over to ensure regulated control close tospecifications. To enable the use of one fuzzy controller instance overthe entire dynamic range of flow operating setpoints, the combinedcontroller input and output sets are scaled when flow specificationsdiffer from the original calibrated setting.

Thus, in an embodiment the controller 22 is configured to automaticallyswitch between pressure control and flow rate control during one or moreof the steps of the analysis cycle. In the pressure control mode thecontroller 22 regulates the speed of the respective syringe pump 10,12by controlling the respective stepper motor 14,16 with the aim tomaintain a desired pressure downstream of the pump concerned. Thedesired pressure may vary from step to step or during a step of thecycle. In the flow rate control mode the controller 22 regulates thespeed of the respective syringe pump 10,12 by controlling the respectivestepper motor 14,16 with the aim to maintain a desired flow ratedownstream of the pump. The desired flow rate may vary from step to stepor during a step of the cycle.

In another embodiment, the controller is configured to automaticallyswitch between the pressure and flow control rate in one or more stepsof the cycle on the basis of fuzzy logic.

During the elution and separation step in the flow rates of the pumps10,12 needs to follow in exactly predefined profile, and automaticchanging between pressure and flow control would not be useful in thisstep. However in the other steps of the cycle, significantly higher flowrates can be achieved by applying automatic switching between pressurecontrol and flowrate control. Increased flow rates can be achieved inthe steps where the automatic change between the control modes isapplied and thereby the overall analysis cycle time can be reduced. Anincreasing advantage of such optimized switching between pressurecontrol and flowrate control is obtained with decreasing cross-sectionalarea of the various columns and conduits in the high-performance liquidchromatography device 1. However, for steps in which the fluid does notneed to be urged to the column 50, an increase in diameter of theconduits of the device will allow even further increased flow rates thatalso will be able to be exploited by a fuzzy logic-based controller andresult in even further reduce its overall cycle times.

The various aspects of what is described above can be used alone or invarious combinations. The teaching of this application is preferablyimplemented by a combination of hardware and software, but can also beimplemented in hardware or software. The teaching of this applicationcan also be embodied as computer readable code on a computer readablemedium, i.e. a computer readable medium including at least computerprogram code for controlling the operation of a pump and program codefor automatically switching between a pressure control mode and a flowrate control mode. In an embodiment the computer readable mediumincludes program code for applying fuzzy logic.

It should be noted that the teaching of this application is not limitedto the use of a single controller or to a device with the exactconfiguration as described above.

The teaching of this application has numerous advantages. Differentembodiments or implementations may yield one or more of the followingadvantages. It should be noted that this is not an exhaustive list andthere may be other advantages which are not described herein. Oneadvantage of the teaching of this application is that it provides for amethod of controlling a pump of a liquid chromatography apparatus andfor a liquid chromatography apparatus with an optimized analysis speed.Another advantage of the teaching of this application is that itprovides for a method of controlling a pump of a liquid chromatographyapparatus and a chromatography apparatus with an improved ease of use.Yet another advantage of the teaching of this application is that itprovides for a method for controlling the pump of a chromatographyapparatus and a chromatography apparatus with improved likelihood offlawless analysis execution.

Although the teaching of this application as been described in detailfor purpose of illustration, it is understood that such detail is solelyfor that purpose, and variations can be made therein by those skilled inthe art without departing from the scope of the teaching of thisapplication.

For example, although the teaching of this application has beendescribed in terms of a high-performance liquid chromatography apparatuswith a syringe pump, it should be appreciated that the invention mayalso be applied to other types of pumps, such as piston pumps,reciprocating pumps, pneumatic pumps, hydraulic pumps, piezo-drivenpumps, electrokinetic pumps, peristaltic pumps and the like. It shouldalso be noted that there are many alternative ways of implementing themethods and apparatuses of the teaching of this application. Forexample, although the pump controller has been described in terms of adedicated control unit, it should be noted that other controllers can beused. For example, a general-purpose computer or the like may be used insome configurations of the liquid chromatography apparatus.

The term “comprising” as used in the claims does not exclude otherelements or steps. The term “a” or “an” as used in the claims does notexclude a plurality. The single processor or other unit may fulfill thefunctions of several means recited in the claims.

1.-20. (canceled)
 21. A method for controlling fluid flow in a liquidchromatography apparatus, said method comprising: driving said flow witha pump, controlling said fluid flow by either: measuring the pressure ofsaid liquid and in response thereto adjusting the output of said pump toachieve a target fluid pressure, or measuring the fluid flow of saidliquid and in response thereto adjusting the output of said pump toachieve a target fluid pressure, and automatically switching orbalancing between pressure based fluid flow control and flow rate basedfluid flow control, said method further comprising involving fuzzy logicalgorithms for deciding when to switch or how to balance between saidpressure based fluid flow control and flow rate based fluid control. 22.A method according to claim 21, wherein said target fluid pressureand/or said target flow rate are variable during the execution of themethod.
 23. A method according to claim 21, wherein said liquidchromatography apparatus is operated in an analysis cycle that involvesa plurality of chronological steps that have different fluid flowrequirements, and one or more of said steps being predetermined to beoperated with either flow rate based control or pressure based control.24. A method according to claim 23, wherein one or one or more of saidsteps are not predetermined to be operated with a particular one of theflow rate based control or pressure based control, and the control modein these steps is determined on the basis of measured parametersrelating to operation of the liquid chromatography apparatus.
 25. Amethod according to claim 23, wherein the regulatory output is afunction of the time point relative to the overall analysis cycle of theapparatus.
 26. A method according to claim 25, wherein the regulatoryoutput is different between steps and can be modified with theprogression within each step.
 27. A method according to claim 22,wherein the fuzzy logic has the current flow rate and the currentpressure downstream of the pump as the input parameters.
 28. A methodaccording to claim 22, wherein the fuzzy logic output parameter is thepump speed.
 29. A method according to claim 22, wherein said fuzzy logicis arranged and divided into a plurality of overlapping fuzzy sets. 30.A method according to claim 29, wherein said fuzzy logic sets areconfigured to give both course- and fine-grain regulationcharacteristics.
 31. A method according to any of claim 22, wherein theoutput variable of the fuzzy logic is arranged into a plurality ofdiscrete singleton outputs, which are subsequently defuzzified into acontrol value by a center of gravity method.
 32. A method according toclaim 22, wherein the fuzzy variables are featured in a plurality ofrules, aiming to optimize flow, while at the same time ensuring that amaximum pressure is not exceeded.
 33. A method according to claim 32,wherein rules pertaining to pressure regulation are prioritized by rulefactors, so that pressure regulation will take precedence over any flowoptimization, when the regulator operates in that section of the inputspace.
 34. A method according to claim 22, wherein the fuzzy logic flowcontrol comprises an iterating loop that takes the sensor readouts,supplies them to a fuzzy logic engine and has the fuzzy logic enginere-evaluate its flow output.
 35. A method according to claim 22, whereinthe output difference is calculated relative to the previous outputvalue and said difference is clamped into a specific range, added to theprevious output value, and supplied to the pump as a new pump speed. 36.A liquid chromatography apparatus comprising: at least one pump, a flowrate sensor downstream of said pump, a pressure sensor downstream ofsaid pump, and a pump controller coupled to said pump, to said flow ratesensor and to said pressure sensor, said pump controller beingconfigured to automatically switch between or balance pressure basedpump control and flow rate based pump control.
 37. A liquidchromatography apparatus according to claim 36, wherein said pumpcontroller includes a fuzzy logic engine.
 38. A computer readable mediumincluding at least computer program code for controlling the operationof a pump and program code for automatically switching between orbalancing a pressure control mode and a flow rate control mode.
 39. Acomputer readable medium according to claim 38, further includingprogram code for applying fuzzy logic.